이 작품은 NIDDK KO1 DK075386 부여하고 Jun Sun에 미국 암 협회 RSG - 09 - 075-01 - MBC에 의해 지원되었다.

세균성 문화, 마우스 gavage 및 살모넬라균 검출이 프로토콜은 세 부분을 포함하고 있습니다. 1. 살모넬라균의 성장 상태. <ol><li> ° C 야간 37 품어 살모넬라균 Luria - Bertani 국물 (LB) 접시를 준비합니다. </li><li> LB 판에서 복제를 선택하고 12 ML 튜브 7 ML의 LB에 넣어 37 ° C 약 5 시간 동안 흔들. </li><li> 약 18 시간 동안 떨지 않고 37 ° C에서 고정 상 문화와 부화의 0.05 ML과 함께 50 ML의 LB의 예방. </li><li> 비율 100:3을 (HBSS LB)를 사용하여 HBSS에있는 박테리아를 중단, 10 분 6,000 RPM과 함께 실온에서 하룻밤 박테리아 문화를 스핀. 예를 들면 다음과 같습니다 모든 50 ML의 LB 문화는 1.5 ML의 HBSS에 일시 중지됩니다. </li><li> 동물 gavage 경우, 추가 1시 10분 비율 박테리아 문화를 희석. 예를 들면 다음과 같습니다 모든 1.5 ML의 LB 문화 15 ML의 HBSS에 일시 중지됩니다. </li></ol> 2. 살모넬라균 감염 모델 * <ol><li> 스트렙토 마이신 솔루션을 준비합니다. 각각의 마우스는 100 μl HBSS에 스트렙토 마이신의 7.5 MG를 받게 될 것입니다. 예를 들어, 10 마우스에 대한 1.2 ML HBSS에 스트렙토 마이신 총 90 MG를 준비합니다. 스트렙토 마이신 솔루션을 준비할 때 항상 몇 가지 여분의 볼륨을했습니다. </li><li> 구두 gavage 치료를 받기 전에 물과 음식을 4 시간 철수. </li><li> 스트렙토 마이신 (제어 마우스에 대한 HBSS 100 μl)의 7.5 MG로 스트렙토 마이신과 Gavage 쥐. 똑바로 식도를 만들기 위해 검지로 머리와 목 스트레칭, 단단히 엄지와 가운데 손가락으로 마우스 어깨 위에 피부를 잡아. 입천장을 따라와 인두의 뒤쪽의 오른쪽 방향으로 먹이 바늘의 볼 - 팁을 직접 다음, 부드럽게 식도로 아래로 통과하고 100 μl 솔루션을 주입. 아무 저항도 느끼지해야합니다. </li><li> 스트렙토 마이신 처리, 철회 물, 생쥐는 박테리아에 감염된 음식을 다시하기 전에 이후 20 시에. </li><li> HBSS 100 μl 현탁액 각 마우스를 Gavage 또는 경구 gavage에 의해 멸균 HBSS (제어)와 치료. gavage 절차는 스트렙토 마이신과 같은) 2.3로 gavage 마우스입니다. </li></ol> * 동물 실험은 특정 - 병원체가없는 여성 C57BL / 이전 1 설명된대로 6~7주 살 때 6 쥐 (타코닉, 허드슨, NY)를 사용하여 수행되었습니다. 프로토콜은 동물 자원 (UCAR)에서 로체스터 대학위원회의 대학에 의해 승인되었습니다. 3. 소장에서 살모넬라균의 검출. <ol><li> (100 MG) 마우스 배설물 수집합니다. </li><li> 적극적으로 1ml PBS와 소용돌이와 1.5 마이크로 원심 튜브에 배설물 샘플을 전송합니다. </li><li> 800 RPM 10 분 원심 분리기. 깨끗한 microfuge 튜브에 뜨는을 전송합니다. </li><li> 5 분을 위해 6,000 rpm으로 원심 분리. 뜨는은 삭제됩니다 200 μL PBS는 쥐똥에 추가 vortexed됩니다. </li><li> 살모넬라균을 검출 BBL CHROMagar 접시에 일회용 휴대 확장기와 리크 200 μl PBS가. 살모넬라균 종은 (보라색으로 장미, Fig.1 참조) 자주빛 나타납니다. 200 μl 산출하면 접시에 너무 많은 식민지는 100 μl 또는 연속 50 μl를 사용합니다. </li></ol> 4. 대표 결과. 프로토콜이 제대로되면 살모넬라균 typhimurium의 식민지가 6 개월 이상 마우스 소장에서 검색된 수 있습니다. 살모넬라균은 6 개월 (Fig.1)을 통해 지저분한 문화에 의해 감지 수 있습니다. 전형적인 아웃이 모델의 결과로서, 몸은 상실과 죽음 4 주 이후에 감염 내에서 발생하는 무게. 감염에 사용되는 살모넬라균의 변종에 따라, 일부 마우스는 6 개월 이상 생존할 수 없습니다. <img src="/files/ftp_upload/1947/1947fig1.jpg" alt="그림 1" /> 그림 1. 살모넬라균의 종의 창자 살모넬라균 인해 선택 chromogens의 존재에 신진 대사의 차이로 색상 (퍼플 로즈) 자주빛 나타납니다. 다른 박테리아가 어느 저해 또는 파란색 - 녹색 또는 무색의 식민지를 생산하고 있습니다. <img src="/files/ftp_upload/1947/1947fig2.jpg" alt="그림 2" /> 그림 2. 살모넬라균 돌연변이 스트레인 PhoP C, C PhoP에 감염된 마우스의 생존 비율 아브라 -, 그리고 PhoP C AvrA-/AvrA +, 사주 (28 일) PhoP C 아브라. 4 주 동안 살모넬라균에 감염된 생쥐의 표 1. 체중. <table border="1"><tbody><tr><td></td><td> 0 </td><td> 일주 </td><td> 이주 </td><td> 삼주 </td><td> 사주 </td></tr><tr><td> 제어 </td><td> 16.78 ± 1.05 </td><td> 16.91 ± 1.28 </td><td> 18.26 ± 1.23 </td><td> 19.31 ± 1.26 </td><td> 20.26 ± 1.15 </td></tr><tr><td> PhoP C </TD&gt; <td> 16.89 ± 1.03 </td><td> 17.14 ± 1.19 </td><td> 17.43 ± 1.63 * </td><td> 18.68 ± 1.78 </td><td> 20.05 ± 1.11 </td></tr><tr><td> PhoP C 아브라 - </td><td> 16.91 ± 1.12 </td><td> 16.96 ± 1.39 </td><td> 17.06 ± 2.14 ** </td><td> 18.71 ± 2.18 </td><td> 20.15 ± 1.56 </td></tr><tr><td> PhoP C AvrA-/AvrA + </td><td> 16.94 ± 0.96 </td><td> 17.17 ± 1.02 </td><td> 17.63 ± 1.42 * </td><td> 18.44 ± 2.03 </td><td> 20.09 ± 1.17 </td></tr></tbody></table> * 그룹 P에게 &lt;0.05를 제어 비교 ** 그룹 P &lt;0.01를 제어 비교 표 2. 살모넬라균의 변종이 연구에 사용. <table border="1"><tbody><tr><td> 이름 </td><td> 설명 </td><td> 참조 또는 소스 </td></tr><tr><td> 살모넬라균 14028s </td><td> 야생 형 S. typhimurium </td><td> ATCC </td></tr><tr><td> PhoP C </td><td> 돌연변이 비 병원성 복잡한 조절기 </td><td> 밀러 외., 1990 </td></tr><tr><td> PhoP C 아브라 - </td><td> 아브라 - 돌연변이 </td><td> 콜리 - 하이엄스 외., 2002 </td></tr><tr><td> PhoP C AvrA-/AvrA + </td><td> PhoP C 보완 플라스미드 인코딩 아브라 ​​아브라 -로 </td><td> 콜리 - 하이엄스 외., 2002 </td></tr></tbody></table>

<ol>
	<li>Duan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Catenin+activity+negatively+regulates+bacteria-induced+inflammation.">beta-Catenin activity negatively regulates bacteria-induced inflammation.</a> Lab Invest. 87 (6), 613-624 (2007).</li><li>Barthel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+of+mice+with+streptomycin+provides+a+Salmonella+enterica+serovar+Typhimurium+colitis+model+that+allows+analysis+of+both+pathogen+and+host.">Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.</a> Infection and immunity. 71 (5), 2839-2858 (2003).</li><li>Miller, S. I., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+expression+of+the+phoP+regulon+attenuates+Salmonella+virulence+and+survival+within+macrophages.">Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages.</a> Journal of bacteriology. 172 (5), 2485-2490 (1990).</li><li>Valdez, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nramp1+expression+by+dendritic+cells+modulates+inflammatory+responses+during+Salmonella+Typhimurium+infection.">Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection.</a> Cell Microbiol. 10 (8), 1646-1661 (2008).</li><li>Woo, H., Okamoto, S., Guiney, D., Gunn, J. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions:+Host+resistance+factor+Nramp1+up-regulates+the+expression+of+Salmonella+pathogenicity+island-2+virulence+genes.">Host-pathogen interactions: Host resistance factor Nramp1 up-regulates the expression of Salmonella pathogenicity island-2 virulence genes.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 15705-15710 (2002).</li><li>Townsend, P., Morton, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Animal+Care+Policies+and+Regulations:+United+Kingdom.">Laboratory Animal Care Policies and Regulations: United Kingdom.</a> ILAR J. 37 (2), 68-74 (1995).</li><li>Olfert, E. D., Godson, D. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+enteric+salmonella+infection+in+mice+leads+to+severe+and+persiste+nt+intestinal+fibrosis.">Chronic enteric salmonella infection in mice leads to severe and persiste nt intestinal fibrosis.</a> Gastroenterology. 134 (3), 768-780 (2008).</li><li>Sukupolvi, S., Edelstein, A., Rhen, M., Normark, S. J., Pfeifer, J. 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관심 없음 충돌 선언하지 않습니다.

이 시스템을 사용하려면, 그것은 동물을 gavage하는 방법을 알아보려면 연구자가 필요합니다. 박테리아 문화를 준비하고 쥐를 gavage 위해 우리는 세부 방법론을. 우리는 또한 위장 (GI) 트랙트에서 살모넬라균 지속성을 모니터링하는 방법을 보여줍니다. 포함이 프로토콜의 중요한 단계 <ol><li> 스트렙토 마이신 - 전처리 : 스트렙토 마이신 - 전처리 일부 공생 굿 식물 제거 및 살모넬라균 ?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>ABCD1234</td>
<td>&nbsp;</td>
<tr>
<td>Luria-Bertani broth</td>
<td>&nbsp;</td>
<td>BD</td>
<td>244610</td>
<td>&nbsp;</td>
<tr>
<td>Feeding needle</td>
<td>&nbsp;</td>
<td>Popper &amp; Sons, Inc</td>
<td>7920</td>
<td>&nbsp;</td>
<tr>
<td>Streptomycin </td>
<td>&nbsp;</td>
<td>MP biomedicals, LLC</td>
<td>100556</td>
<td>&nbsp;</td>
<tr>
<td>BBL CHROMagar Salmonella</td>
<td>&nbsp;</td>
<td>BD</td>
<td>214983</td>
<td>&nbsp;</td>
<tr>
<td>Disposable cell spreaders</td>
<td>&nbsp;</td>
<td>Biologix research company</td>
<td>65-1010</td>
<td>&nbsp;</td>		</tbody>
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		</div>

chronic salmonella infected mouse model

세균성 감염 마우스 모델은 감염, 염증, 면역학, 신호 전달, 그리고 tumorigenesis과 같은 분야를 공부를위한 강력한 모델 시스템입니다. 많은 연구자에 의해 유도된 대장염의 이점을 데리고<em&gt; 살모넬라균</em염증과 감염의 초기 단계에있는 연구&gt; typhimurium. 단, 몇 가지 보고서는 만성 감염에<em&gt; 생체내에</em&gt;. 와 쥐<em&gt; 살모넬라균</em위장관에서&gt; 지속적인 존재는 우리가 장기 호스트 세균 상호 작용, 신호 전달, 그리고 tumorigenesis를 탐험하실 수 있습니다. 우리는 함께 만성 세균 감염 마우스 모델을 설립<em&gt; 살모넬라균</em6 개월 이상 마우스 소장에서&gt; typhimurium의 식민지. 이 시스템을 사용하려면, 그것은 박테리아 문화를 준비하고 동물을 gavage하는 방법을 알아보려면 연구자가 필요합니다. 준비 박테리아 문화와 gavage 생쥐에 대한 우리는 세부 방법론을. 우리는 또한을 감지하는 방법을 보여줍니다<em&gt; 살모넬라균</em위장관에서&gt; 끈기. 전반적으로,이 프로토콜은 기본적으로 중요한 생물 학적 및 미생물 질문을 주소로 세균성 감염 마우스 모델을 사용하여 연구를 지원합니다.

Watch this Scientific Journal Video about Chronic Salmonella Infected Mouse Model at JoVE.com

Chronic Salmonella Infected Mouse Model

영구와 만성 세균 감염 마우스 모델 수립<em&gt; 살모넬라균</em27주에 대한 소장에서&gt; typhimurium의 식민지.

만성의 살모넬라균 감염된 마우스 모델

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

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Biology

Chronic Salmonella Infected Mouse Model

17.2K 조회수.  University of Rochester. 영구와 만성 세균 감염 마우스 모델 수립 살모넬라균 typhimurium의 식민지.

비디오: Chronic Salmonella Infected Mouse Model

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

대장암의 Orthotopic 마우스 모델

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

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생물학

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

SD 쥐에서 레이저 유도 만성 안구 고혈압 모델

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

형광 항체를 사용하여 마우스 표피의 파라핀 섹션에 Immunohistochemistry

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Murine 콜린 불충분 한, Ethionine 보충 (CDE) 다이어트 만성 간 상해의 모델

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

트랜스-내부 세포 배아 줄기 세포의 대량 주입 리드 더 높은 Chimerism 요금

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

장 부분 방해의 마우스 모델

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

음식과 환경 샘플에서 살 모 넬 라 의 준 metagenomic 분석

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.